Molecular cloning, expression, and partial characterization of two novel members of the ovalbumin family of serine proteinase inhibitors.

A human placental lambda gt11 cDNA library was screened for sequences encoding proteins related to human proteinase inhibitor 6 (PI6), and two plaques were identified that displayed weak hybridization at high stringency. Isolation and characterization of the DNA inserts revealed two novel sequences encoding proteins composed of 376 and 374 amino acids with predicted molecular masses of approximately 42 kDa. The novel proteins displayed all of the structural features unique to the ovalbumin family of intracellular serpins including the apparent absence of a cleavable N-terminal signal sequence. The degree of amino acid sequence identity between the novel serpins and PI6 (63-68%) significantly exceeds that of any other combination of known intracellular serpins. The two novel serpins encoded by the two novel cDNA sequences have been designated as proteinase inhibitor 8 (PI8) and proteinase inhibitor 9 (PI9). The putative reactive center P1-P1' residues for PI8 and PI9 were identified as Arg339-Cys340 and Glu340-Cys341, respectively. PI9 appears to be unique in that it is the first human serpin identified with an acidic residue in the reactive center P1 position. In addition, the reactive center loop of PI9 exhibits 54% identity with residues found in the reactive center loop of the cowpox virus CrmA serpin. Two PI8 transcripts of 1.4 kilobases (kb) and 3.8 kb were detected by Northern analysis in equal and greatest abundance in liver and lung, while the 1.4-kb mRNA was in excess over the 3.8-kb mRNA in skeletal muscle and heart. Two PI9 transcripts of 3.4 and 4.4 kb were detected in equal and greatest abundance in lung and placenta and were weakly detected in all other tissues. PI8 and PI9 were expressed in baby hamster kidney and yeast cells, respectively. Immunoblot analyses using rabbit anti-PI6 IgG indicated the presence of PI8 in the cytosolic fraction of stably transfected cells that formed an SDS-stable 67-kDa complex with human thrombin. PI9 was purified to homogeneity from the yeast cell lysate by a combination of heparin-agarose chromatography and Mono Q fast protein liquid chromatography and migrated as a single band in SDS-polyacrylamide gel electrophoresis with an apparent molecular mass of 42 kDa. Purified recombinant PI9 failed to inhibit the amidolytic activities of trypsin, papain, thrombin, or Staphylococcus aureus endoproteinase Glu-C and did not form an SDS-stable complex when incubated with thrombin. The cognate intracellular proteinases that interact with PI8 and PI9 are unknown.

Elucidation of the mechanism of CryIIIA overproduction in a mutagenized strain of Bacillus thuringiensis var. tenebrionis.

NB176 is a Bacillus thuringiensis mutant derived by gamma-irradiation of NB125 Bacillus thuringiensis var. tenebrionis (Krieg). It exhibits two interesting phenotypes: (i) oligosporogeny and (ii) twofold to threefold overproduction of the CryIIIA protein. Southern profiles of the NB176 strain showed an additional copy(s) of the cryIIIA gene located on a 4 kb HindIII fragment, in addition to the expected cryIIIA gene on a 3 kb HindIII fragment. Each cryIIIA gene-bearing HindIII fragment was cloned from NB176. The restriction map of the 3 kb HindIII fragment was identical to that published by Donovan and coworkers. Sequencing of the 4 kb HindIII fragment showed no alterations in the promoter region of the cryIIIA gene but did show replacement of the region immediately following the cryIIIA open reading frame with a sequence encoding a transposase with 50% amino acid homology to that of Tn1000. These findings suggest that the overproduction phenotype of NB176 results from extra copies of the cryIIIA gene produced from a transposition event(s) induced or stabilized by gamma-irradiation. Integration of additional copies of the cryIIIA gene into the native 90 MDa plasmid of the wild-type B. thuringiensis var. tenebrionis strain resulted in strains that made enormous crystals, many possessing greatly enhanced insecticidal activity.

Molecular cloning and characterization of a novel transglutaminase cDNA from a human prostate cDNA library.

The primary sequence of a cDNA encoding a novel transglutaminase from a human prostate cDNA library is reported. The sequence is compared to other known transglutaminases in a multiple alignment. The deduced peptide sequence is 51% identical to a rat prostate transglutaminase.

Molecular cloning, expression, and partial characterization of a second human tissue-factor-pathway inhibitor.

Previous studies have shown that tissue-factor-pathway inhibitor (TFPI) is an important regulator of the extrinsic pathway of blood coagulation through its ability to inhibit factor Xa and factor VIIa-tissue factor activity. We describe the molecular cloning and expression of a full-length cDNA that encodes a molecule, designated TFPI-2, that has a similar overall domain organization and considerable primary amino acid sequence homology to TFPI. After a 22-residue signal peptide, the mature protein contains 213 amino acids with 18 cysteines and two canonical N-linked glycosylation sites. The deduced sequence of mature TFPI-2 revealed a short acidic amino-terminal region, three tandem Kunitz-type domains, and a carboxyl-terminal tail highly enriched in basic amino acids. Northern analysis indicates that TFPI-2 is transcribed in umbilical vein endothelial cells, liver, and placenta. TFPI-2 was expressed in baby hamster kidney cells and purified from the serum-free conditioned medium by a combination of heparin-agarose chromatography, Mono Q FPLC, Mono S FPLC, and Superose 12 FPLC. Purified TFPI-2 migrated as a single band in SDS/PAGE and exhibited a molecular mass of 32 kDa in the presence and absence of reducing agent. The amino-terminal sequence of recombinant TFPI-2 was identical to that predicted from the cDNA. Despite its structural similarity to TFPI, the purified recombinant TFPI-2 failed to react with polyclonal anti-TFPI IgG. Preliminary studies indicated that purified recombinant TFPI-2 strongly inhibited the amidolytic activities of trypsin and the factor VIIa-tissue factor complex. In addition, the inhibition of factor VIIa-tissue factor amidolytic activity by recombinant TFPI-2 was markedly enhanced in the presence of heparin. TFPI-2 at high concentrations weakly inhibited the amidolytic activity of human factor Xa, but had no measurable effect on the amidolytic activity of human thrombin.

The human glucagon receptor encoding gene: structure, cDNA sequence and chromosomal localization.

Characterization of the human glucagon-receptor-encoding gene (GGR) should provide a greater understanding of blood glucose regulation and may reveal a genetic basis for the pathogenesis of diabetes. A cDNA encoding a complete functional human glucagon receptor (GGR) was isolated from a liver cDNA library by a combination of polymerase chain reaction and colony hybridization. The cDNA encodes a receptor protein with 80% identity to rat GGR that binds [125I]glucagon and transduces a signal leading to increases in the concentration of intracellular cyclic adenosine 3',5'-monophosphate. Southern blot analysis of human DNA reveals a hybridization pattern consistent with a single GGR locus. In situ hybridization to metaphase chromosome preparations maps the GGR locus to chromosome 17q25. Analysis of the genomic sequence shows that the coding region spans over 5.5 kb and is interrupted by 12 introns.

Isolation of glucagon antagonists by random molecular mutagenesis and screening.

Glucagon has an important role in the regulation of glucose homeostasis, and glucagon antagonists may be effective therapeutic agents in the control of diabetes mellitus. We were able to identify a number of analogs with antagonist activity by creating libraries of mutant glucagon coding sequences, expressing them in a yeast (Saccharomyces cerevisiae) secretion system, and screening for clones that produce analogs that inhibit the glucagon stimulation of rat hepatocyte membrane adenylate cyclase. These libraries were constructed by allowing random misincorporation during the synthesis of oligonucleotides that contained the complete coding sequence for mammalian glucagon or for an analog (desHis1-glucagon) that had partial antagonist activity. We developed and used a simplified screening assay to test culture broths from > 3500 individual transformant yeast clones for their ability to inhibit glucagon-dependent adenylate cyclase activity. Ultimately, > 20 different analogs with antagonist activity were identified by recovering and sequencing plasmid DNA from yeast strains that were positive in the screening assay. Interestingly, several analogs were identified repeatedly in independent yeast clones and certain amino acid substitutions occurred in more than one analog. This clustering of randomly isolated mutations clearly delineates the regions of the glucagon molecule that are important for designing improved glucagon antagonists. A subset of the antagonists identified in yeast broth were produced by peptide synthesis to confirm their activities as pure compounds.

Molecular cloning, cDNA sequence, and bacterial expression of human glutamine:fructose-6-phosphate amidotransferase.

Glutamine:fructose-6-phosphate amidotransferase (GFAT) has recently been shown to be an insulin-regulated enzyme that plays a key role in the induction of insulin resistance in cultured cells. As a first step in understanding the molecular regulation of this enzyme the human form of this enzyme has been cloned and the functional protein has been expressed in Escherichia coli. A 3.1-kilobase cDNA was isolated which contains the complete coding region of 681 amino acids. Expression of the cDNA in E. coli produced a protein of approximately 77 kDa and increased GFAT activity 4.5-fold over endogenous bacterial levels. Recombinant GFAT activity was inhibited 51% by UDP-GlcNAc whereas bacterial GFAT activity was insensitive to inhibition by UDP-GlcNAc. On the basis of these results we conclude that: 1) functional human GFAT protein was expressed, and 2) the cloned human cDNA encodes both the catalytic and regulatory domains of GFAT since the recombinant GFAT was sensitive to UDP-GlcNAc. Overall, the development of cloned GFAT molecular probes should provide new insights into the development of insulin resistance by allowing quantitation of GFAT mRNA levels in pathophysiological states such as non-insulin-dependent diabetes mellitus and obesity.

Molecular cloning of porcine alveolar macrophage-derived neutrophil chemotactic factors I and II; identification of porcine IL-8 and another intercrine-alpha protein.

Alveolar macrophages (AM) mediate lung inflammation by producing lipid and peptide molecules that attract neutrophils (PMN) to the lung. Recently we described two porcine proteins called alveolar macrophage-derived chemotactic factors, AMCF-I and -II, that are potent, efficacious, and specific PMN chemoattractants both in vitro and in vivo. We report here the cloning of the full-length cDNAs which code for each protein. Porcine AM were stimulated for 4 h in vitro with Escherichia coli endotoxin (LPS), and a cDNA library was created from poly(A)(+)-selected mRNA. Specific oligonucleotide probes for AMCF-I and AMCF-II were amplified from the porcine AM cDNA library by the polymerase chain reaction using degenerate oligonucleotide primer pairs derived from the N-terminal amino acid sequences of the proteins. These probes were used to isolate 2 full-length cDNAs of 1466 (AMCF-I) and 1515 (AMCF-II) base pairs. Both cDNAs code for proteins with four cysteine residues containing the C-X-C sequence characteristic of the intercrine-alpha family of neutrophil chemoattractants. AMCF-I shares 74% identity with human IL-8 and 84% identity with rabbit IL-8, and likely represents the porcine homologue of IL-8. By contrast, AMCF-II has no obvious human homologue. AMCF-II shares 53% identity with human neutrophil activating peptide 2. Its shared identity with the GRO-related proteins is as high as 61% (rat CINC/GRO), and its shared identity with the 78 amino acid epithelial cell-derived neutrophil activator (ENA-78) is 67%. AMCF-II may represent a new member of the intercrine-alpha family of neutrophil chemoattractants.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequence of human galanin and its inhibition of glucose-stimulated insulin secretion from RIN cells.

Human progalanin cDNA was cloned with polymerase chain reaction techniques. The cDNA sequence predicts that the human form of galanin has a substitution of the glycine residue found at position 30 in other species and thus is likely to retain this residue during posttranslational processing and not be amidated at the COOH terminus. Furthermore, the cDNA sequence predicts three additional amino acid substitutions compared with known galanins. Human galanin was synthesized, and its bioactivity was compared with porcine and rat galanin based on inhibition of insulin release from a glucose-responsive rat insulinoma (RIN) cell line. Human galanin inhibited glucose-stimulated insulin secretion in a dose-dependent manner in RIN cells. Human, porcine, and rat galanin exhibited similar activity with ED50 less than 1 nM.

Selection of secretory protein-encoding genes by fusion with PHO5 in Saccharomyces cerevisiae.

Secretory protein-encoding genes of Saccharomyces cerevisiae have been cloned by a novel procedure that is based on the functional selection of their fusions with acid phosphatase (APase) at the DNA level. DNA fragments that functionally replace the promoter and signal sequence-encoding regions of the PHO5 gene (encoding APase) have been obtained by positive selection from a pool of cloned random DNA fragments. Five unique DNA sequences containing the promoter, and encoding signal sequences have been isolated. We have also isolated the complete gene, SSP120, encoding one of these S. cerevisiae secretory proteins, SSP120. Gene disruption studies have shown that the SSP120 gene is not essential for viability and growth. The SSP120 amino acid (aa) sequence has 13.5% identity with the middle 88-250 aa residues of the chicken glycosylation site-binding protein. However, SSP120 disruption did not affect protein glycosylation in yeast. The present study provides an alternative approach for the isolation of genes encoding secretory proteins, in contrast to classical genetic approaches that require isolation of functionally defective mutations followed by gene isolation by functional complementation. The present procedure should contribute to our understanding of protein sorting by permitting the cloning of genes encoding proteins targeted to different organelles in the secretory pathway.

Cloning and primary structure of a human islet isoform of glutamic acid decarboxylase from chromosome 10.

Glutamic acid decarboxylase (GAD; glutamate decarboxylase, L-glutamate 1-carboxy-lyase, EC 4.1.1.15), which catalyzes formation of gamma-aminobutyric acid from L-glutamic acid, is detectable in different isoforms with distinct electrophoretic and kinetic characteristics. GAD has also been implicated as an autoantigen in the vastly differing autoimmune disease stiff-man syndrome and insulin-dependent diabetes mellitus. Despite the differing GAD isoforms, only one type of GAD cDNA (GAD-1), localized to a syntenic region of chromosome 2, has been isolated from rat, mouse, and cat. Using sequence information from GAD-1 to screen a human pancreatic islet cDNA library, we describe the isolation of an additional GAD cDNA (GAD-2), which was mapped to the short arm of human chromosome 10. Genomic Southern blotting with GAD-2 demonstrated a hybridization pattern different from that detected by GAD-1. GAD-2 recognizes a 5.6-kilobase transcript in both islets and brain, in contrast to GAD-1, which detects a 3.7-kilobase transcript in brain only. The deduced 585-amino acid sequence coded for by GAD-2 shows less than 65% identity to previously published, highly conserved GAD-1 brain sequences, which show greater than 96% deduced amino acid sequence homology among the three species. The function of this additional islet GAD isoform and its importance as an autoantigen in insulin-dependent diabetes remain to be determined.